Plants generally exhibit an antibiotic reaction (hypersensitive reaction) when they come into contact with a pathogenic fungi, and are known to produce phytoalexins having antifungal activity against the pathogenic fungus in the tissue around the reaction site. Examples of phytoalexins include momilactones A and B, oryzalexins A, B, C, D, E, F, and S, sakuranetin, oryzalic acids A and B, oryzalides A and B, and phytocassanes A, B, C, and D (Japanese Patent Application No. 7-43520/1995) discovered by the present inventors.
Substances that induce phytoalexin production in plant bodies are referred to as elicitors (N. T. Keen, Science 187: 74-75 (1975)), most of which have thus far been isolated from plant pathogenic fungi. Typical elicitors include hepta-.beta.-D-glucopyranoside as polysaccharide isolated from Phytophthora megasperma f. sp. glycinea (J. K. Sharp, B. Valentand, P. Albersheim, J. Biol. Chem. 259: 11321-11336 (1984)), monicholine A as protein substance isolated from Monilinia fructicola (I. A. M. Cruickshank and D. R. Perrin, Life Sci. 7: 449-458 (1968)), and eicosapentaenoic acid as lipid isolated from Phytophthora infestans (R. M. Bostock, J. Kuc, and R. A. Laine, Science 212: 67-69 (1975)).
Since, as noted above, elicitors have action in inducing the production of phytoalexins in plant bodies that have antifungal activity against pathogenic fungi, they are considered substances which could serve as active ingredients in highly safe agents for controlling plant diseases based on an action different than that of conventional agrochemicals, and there has been a strong need for the discovery of useful elicitors and for novel screening methods that would allow substances having elicitor activity to be rapidly and easily screened as methods for discovering such useful elicitors.
Under these circumstances, the inventors undertook extensive and painstaking research in view of the aforementioned prior art to develop a novel screening method for screening useful elicitors that induce phytoalexin production in rice plants, but it is extremely difficult to screen for substances having elicitor activity using rice plant bodies, so much so that no effective methods have yet been established. In their various studies to develop an effective method, the inventors have succeeded in establishing a new basic technique relating to the species of test rice plants, rice cultivating methods, methods for applying test samples, phytoalexin analyses, and the like, and thus have discovered that substances having elicitor activity can be screened.
That is, as a result of their studies on rice used in tests, they have found that the matters relating to the variety and species of test rice plants, the cultivation temperature and humidity, the age of the test rice plants, the site where the test sample is applied, and the like are extremely important, and they have found that various optimal conditions of the matters can be established for using the same as a screening method. They also have determined a method for extracting phytoalexins induced in rice plant bodies and for analyzing them by HPLC, using phytocassanes and momilactones as preferred examples of phytoalexins.
Tests of various substances by the aforementioned method have revealed a number of compounds having elicitor activity.
The inventors attempted to screen substances having elicitor activity from microbial products by using the aforementioned screening method, and thus have discovered the substances having such elicitor activity widely existing in filamentous fungi, including plant pathogenic fungi.
Further, they also isolated such substances by means of solvent extraction, column chromatography, or the like, to obtain three types of resulting active substances, and structurally analyzed them and thereby they ascertained that the substances were the cerebroside compounds PO8, PO9, and R2 having the following structural formulas, and that these substances were effective as active ingredients in agents for controlling diseases in rice plants, and they have accomplished the present invention. ##STR1##
The chemical structures of the cerebroside compounds PO8, PO9, and R2 which discovered by them as substances having elicitor activity have already reported in references. PO8 has the same structure as cerebroside A (R. D. Sitrin et al, J. Antibiot. 41, 469-480 (1988)), PO9 has the same structure as PENII (G. Kawai et al, Agric. Biol. Chem. 49, 2137-2146 (1985)), and R2 has the same structure as cerebroside B (G. Kawai et al, J. Lipid. Res. 26, 338-343 (1985)). The substances having the elicitor activity related to the present invention, however, have not been described in the references and the inventors have firstly ascertained that these cerebroside compounds have activity in controlling diseases in rice plants.